Immobilization can be defined as association of a substance, being immobilized, with another substance, generally termed a support or carrier, so that the associated (i.e. immobilized) substance retains activity and remains substantially associated during the course of chemical processing in which it may be used. Immobilized biologically active proteins, such as antibodies, antigens, enzymes and the like, are highly useful when the association is long lasting and the activity of the biologically active protein is not significantly decreased by its immobilization. Among the reasons for immobilizing a biologically active protein, a most common one is simply to facilitate its recovery or separation from a product prepared by using the protein. Other reasons include: those of ease of reactivating of the protein, should it become deactivated during use; ability to repeatedly reuse it in subsequent processes so long as adequate activity is retained; ability to employ the immobilized protein for continuous processing; and the like.
The art teaches a variety of manners for immobilization, as well as various substances including crushed red brick, porous ceramics, glass beads, wood chips, paper, polymeric resins, and the like, which serve as the support or carrier. These support or carrier materials generally are insoluble materials in the form of solids or gels to facilitate, such as by filtering, centrifuging, precipitation and the like, their separation along with their associated substances from the processing medium in which they provided their chemical function, such as serving as a catalyst. Art taught immobilizations, include encapsulation of the protein with diffusion of reactants and products through an encapsulating membrane. Other art techniques include: entrapment in polymeric gels; cross-linking with bifunctional agents to provide large enough agglomerates enabling separation from liquid mediums; and most frequently chemical attachment, such as by covalent linkage, to the support or substrate. Another technique is an adsorption of the biologically active protein to a solid support or carrier. Each of the foregoing manners and techniques provides some advantages and suffers from some disadvantages.
Probably the simplest and most economical manner is the last-mentioned of adsorption to a solid support. Most biologically active proteins usually are hydophilic or at most weakly hydrophobic and do not adsorb strongly to the most widely used supports. In the absence of strong adsorption, the adsorbed product is not stable with weakly adsorbed protein being lost, such as washed away and/or carried along with the medium, containing product, and with difficulty of separation of the product therefrom. Improvements in attachment of biologically active proteins to hydrophobic carriers by adsorption and noncovalent interaction are highly desirable.
It is now generally known that hydrophobic "pockets" or "patches" are present on the surfaces of most proteins ("The Hydrophobic Effect", C. Tanford, (1980) Wiley-Interscience, N.Y., N.Y.). Proteins differ in their sizes, shapes and number of such patches. Their affinities for hydrophobic materials, thus, also differ and this has been exploited for their separation and purification from each other by a technique called "hydrophobic chromatography". For proteins with a substantial amount of hydrophobic surface area, adsorption to a hydrophobic support can be very strong, in some cases so strong that they cannot effectively be removed even upon washing with high concentrations of organic solvents (L. G. Butler, "Arch. Biochem. Biophys." 171 (1975), 645-650; K. D. Caldwell et al. "Biotechnol. Bioeng." 17 (1975) 613-616). It has been observed that many enzymes retain all or most of their catalytic activity when adsorbed in this manner and this observation has been exploited to prepare immobilized forms of some of these enzymes (Butler, supra). This approach is limited, however, since most enzymes and other proteins are hydrophilic or weakly hydrophobic and do not adsorb strongly to such supports. Some can be immobilized in this manner but gradually leach away in the course of their use.
In 1981, H. Wu and G. E. Means ("Biotech. Bioengr." 23 (1981), 855-861) described a procedure related to that of this invention but differing in a number of important ways. The type of reagent differs, that is, hydrophobic aldehydes plus NaBH.sub.3 CN, and reactions were conducted in the presence of a support. The present invention overcomes disadvantages of that procedure.
This invention's method differs from those now used for conventional enzyme immobilization. A typical art method involves a step or series of steps to introduce a reactive group onto the support, followed by a step wherein the enzyme and activated support are allowed to react (K. Mosbach, "Methods in Enzymology", 44 (1976), Academic Press, N.Y., N.Y.). The enzyme is attached to the support in such cases, by one or more covalent bonds. The reaction between the soluble protein and a reactive group on an insoluble surface is, however, inherently quite inefficient and this is the source of many problems for such methods. The procedures necessary to introduce a reactive group onto a support are generally expensive and greatly limit the types of support that can be used.
The present invention increases the hydrophobicity of proteins by mild, selective chemical modification in order to strengthen their adsorption onto insoluble hydrophobic surfaces. This approach is applicable to attach a large number of enzymes and other biologically active proteins to a wide variety of hydrophobic materials with the products having important commercial applications.
The present invention involves derivatization of the protein by a relatively inexpensive, soluble reagent. Because both the protein and the reagent are in solution, the reaction is relatively efficient and reaction parameters are easy to control. The modified protein derivative can be isolated and characterized by procedures normally applicable to other soluble proteins. Because the reagents used are analogs of reagents widely used for protein modification, some knowledge exists relative to possible reactions with proteins (G. E. Means and R. E. Feeney, "Chemical Modifications of Protein", (1971), Holden-Day, San Francisco, Calif.). Another step of the procedure involves adsorption to the selected support and resembles procedures described for certain "naturally" hydrophobic proteins.